Substrates, systems and methods for analyzing materials

ABSTRACT

Substrates, systems and methods for analyzing materials that include waveguide arrays disposed upon or within the substrate such that evanescent fields emanating from the waveguides illuminate materials disposed upon or proximal to the surface of the substrate, permitting analysis of such materials. The substrates, systems and methods are used in a variety of analytical operations, including, inter alia, nucleic acid analysis, including hybridization and sequencing analyses, cellular analyses and other molecular analyses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 13/902,032 filed on May24, 2013, which is a continuation of Ser. No. 13/477,865 filed on May22, 2012, now U.S. Pat. No. 8,471,219, which is a continuation of Ser.No. 13/245,578 filed Sep. 26, 2011, now U.S. Pat. No. 8,207,509, whichis a continuation of U.S. patent application Ser. No. 12/896,515, filedOct. 1, 2010, now U.S. Pat. No. 8,053,742, which is a continuation ofU.S. patent application Ser. No. 11/981,795, filed Oct. 31, 2007, nowU.S. Pat. No. 7,834,329, which is a continuation of U.S. patentapplication Ser. No. 11/849,157, filed Aug. 31, 2007, now U.S. Pat. No.7,820,983, which claims the benefit of Provisional U.S. PatentApplication No. 60/841,897, filed Sep. 1, 2006, the full disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

BACKGROUND OF THE INVENTION

A large number of analytical operations benefit from the simultaneousillumination of relatively large area of substrates in order toaccomplish the desired analysis. For example, interrogation ofbiopolymer array substrates typically employs wide area illumination,e.g., in a linearized beam, flood or reciprocating spot operation. Suchillumination allows interrogation of larger numbers of analyticalfeatures, e.g., molecule groups, in order to analyze the interaction ofsuch molecule groups with a sample applied to the array.

In the case of DNA arrays, in particular, large numbers ofoligonucleotide probes are provided in discrete locations on a planarsubstrate surface, such that the surface comprises multiple, smallpatches of identical probes, where the probes' nucleotide sequence foreach patch location is known. When one applies a labeled sample sequenceto the array, the position on the array to which the sample hybridizesis indicative of the complementary probe sequence, and as such, thesequence of the sample sequence. These arrays are generally interrogatedusing laser based fluorescence microscopes that are capable of applyingexcitation illumination over large areas of the substrate in order tointerrogate all of the patches. Such systems have employed galvoscanners, slower, scanning microscopes, linearized beam illumination,and wide area flood illumination.

In some cases, however, a more tightly controlled illumination strategymay be desired. For example, it may be desirable to provide strictercontrol of the volume of material that is illuminated, as well as theoverall area that is illuminated, effectively controlling illuminationnot only in one of the x or y axes of a planar substrate, but also inthe z axis, extending away from the substrate. One example of controlledillumination that accomplishes both lateral (x and y) and volume (z)control is the use of zero mode waveguides as a base substrate foranalyzing materials. See, U.S. Pat. Nos. 6,991,726 and 7,013,054, thefull disclosures of which are hereby incorporated herein by reference intheir entirety for all purposes. Briefly, zero mode waveguide arraysubstrates employ an opaque cladding layer, e.g., aluminum, chromium, orthe like, deposited over a transparent substrate layer, and throughwhich are disposed a series of apertures through to the transparentlayer. Because the apertures are of sufficiently small cross sectionaldimensions, e.g., on the order of 50-200 nm in cross section, theyprevent propagation of light through them that is below a cut-offfrequency. While some light will enter the aperture or core, itsintensity decays exponentially as a function of the distance from theaperture's opening. As a result, a very small volume of the core isactually illuminated with a relevant level of light. Such ZMW arrayshave been illuminated using a number of the methods described herein,including spot illumination, flood illumination and line illumination(using a linearized beam)(See, e.g., co-pending U.S. patent applicationSer. No. 11/483,413 (filed Jul. 5, 2006), and 60/772,908 (filed Feb. 13,2006), the full disclosures of which are incorporated herein byreference in their entirety for all purposes).

While the various foregoing systems and methods have proven some measureof effectiveness, the present invention provides for improvements overthese systems and methods, in a number of respects.

BRIEF SUMMARY OF THE INVENTION

The present invention provides substrates, systems and methods foranalyzing materials. In particular, in at least one aspect, theinvention provides an analytical device that comprises a substratecomprising a first surface and at least a first optical waveguidedisposed upon the first surface. The device includes an analyte disposedsufficiently proximal to the first surface and external to thewaveguide, to be illuminated by an evanescent field emanating from thewaveguide when light is passed through the waveguide, e.g., the lightfield that decays exponentially as a function of distance from thewaveguide surface.

In another aspect, the invention provides a method of illuminating ananalyte that comprises providing a substrate comprising a first surfaceand at least a first optical waveguide disposed upon the first surface.An analyte is provided disposed sufficiently proximal to the firstsurface and external to the waveguide, to be illuminated by anevanescent field emanating from the waveguide when light is passedthrough the waveguide. Light is then directed or propagated through thefirst waveguide such that the evanescent field from the waveguideilluminates the analyte.

In another aspect, the invention provides a system for analyzing ananalyte that comprises a substrate comprising a first surface and atleast a first optical waveguide disposed upon the first surface. Atleast a first light source is provided optically coupled to the at leastfirst waveguide to direct light into the first waveguide. An opticaldetection system is also provided positioned to receive and detectoptical signals from an analyte disposed sufficiently proximal to thefirst surface and external to the waveguide, to be illuminated by anevanescent field emanating from the waveguide when light is passedthrough the waveguide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a Waveguide array used to split lightfrom individual optical fibers into multiple optical fibers.

FIG. 2 schematically illustrates a substrate of the invention.

FIGS. 3A and 3B schematically illustrate alternate configurations of thesubstrates of the invention.

FIG. 4A schematically illustrates a number of waveguide types useful inthe context of the invention. FIG. 4B schematically illustrates theshape of the evanescent electromagnetic field confined by each of thetypes of waveguides.

FIG. 5 schematically illustrates a substrate of the invention withintegrated control elements.

FIG. 6 schematically illustrates a waveguide substrate of the inventionincluding a structural layer disposed over the waveguide surface.

FIG. 7 schematically illustrates illumination of a molecular complexdisposed upon a substrate of the invention.

FIG. 8 schematically illustrates the use of the substrates of theinvention in molecular array based systems.

FIG. 9 schematically illustrates a system of the invention.

DETAILED DESCRIPTION OF THE INVENTION

I. General

The present invention generally provides a simplified method fordelivering illumination to a plurality of discrete analytical regions ona substrate, and does so in a manner that provides better control ofthat illumination. In particular, the present invention providesmethods, systems and substrates that include surface exposed waveguides,such the exponential decay of light outside the waveguide may beexploited in the surface region in the same manner as the light thatenters a ZMW, above, in order to selectively illuminate materialsprovided upon that surface.

The invention provides such substrates alone, as well as in conjunctionwith one or more other components in an overall system, such as reagents(e.g., dyes, enzymes, buffers and other adjuncts, and the like),illumination sources (e.g., lasers, lamps and the like), observation anddetection components or systems (e.g., optical trains including lenses,mirrors, prisms, gratings and the like, detectors such as PMTs,Photodiodes, photodiode arrays, CCDs, EMCCDs, ICCDs, photographic films,and the like).

Also provided are methods and applications of the substrates and systemsof the invention in the illumination and preferably the analysis ofmaterials which more preferably include fluorescent or fluorogenicmaterials. As will be appreciated, the present invention is broadlyapplicable to any application in which one desires to illuminatematerials that are at or proximal to a surface and/or specific locationson a surface, without illuminating materials that are not similarlysituated. Examples of such analyses include illumination, observationand/or analysis of surface bound cells, proteins nucleic acids or othermolecules of interest.

In the context of analysis, the substrates, methods and systems of theinvention impart numerous additional advantages to an analyticaloperation. In particular, because the light of interest is applied in aspatially focused manner, e.g., confined in at least one lateral and oneorthogonal dimension, using efficient optical systems, e.g., fiberoptics and waveguides, it provides a much more efficient use ofillumination, e.g., laser, power. In addition, because illumination isprovided from within confined regions of the substrate itself, issues ofillumination of background or non-relevant regions, e.g., illuminationof non-relevant materials in solutions, auto-fluorescence of substratesand/or other materials, reflection of illumination radiation, aresubstantially reduced. Likewise, this aspect of the invention providesan ability to perform many homogenous assays for which it would begenerally applicable.

In addition to mitigating autofluorescence of the substrate materials,the systems described herein substantially mitigate auto-fluorescenceassociated with the optical train. In particular, in typicalfluorescence spectroscopy, the excitation light is typically directed atthe reaction of interest through at least a portion of the same opticaltrain used to collect the fluorescence, e.g., the objective and othercomponents. As such, autofluorescence of such components will contributeto the detected fluorescence level and provide a fair amount of noise inthe overall detection. Because the systems direct excitation light intothe substrate through a different path, e.g., through an optical fiberoptically coupled to the waveguide in the substrate, thisauto-fluorescence source is eliminated.

Other advantages include an automatic alignment of illumination withanalytes of interest, as such alignment will be self defining, e.g., ananalyte of interest may be that which is provided within a pre-existingillumination region. This level of alignment is also designed (and isthus programmable) and fabricated into the substrate and is thus notprone to any misalignment issues or other sensitivities of otheranalytical systems. As such, the alignment of illumination is highlyrobust.

Finally, the substrates of the invention typically are provided fromrugged materials, e.g., glass, quartz or polymeric materials that havedemonstrated longevity in harsh environments, e.g., extremes of cold,heat, chemical compositions, e.g., high salt, acidic or basicenvironments, vacuum and zero gravity. As such, they provide, ruggedcapabilities for a wide range of applications.

Such illumination is particularly useful in illuminating fluorescentand/or fluorogenic materials upon or proximal to the surface, includingnucleic acid array based methods, substrate coupled nucleic acidsequencing by synthesis methods, antibody/antigen interactions, and avariety of other applications. These and other applications aredescribed below.

II. Substrates

In particularly preferred aspects, a waveguide substrate is used thatprovides for the propagation of a single beam and preferably itsseparation into multiple propagated beams in a waveguide array. Examplesof such arrayed waveguides have been employed in the fiber optic basedsystems in splitting beams propagated by a single fiber into multiplefibers. Typically, however, such arrayed waveguides employ a claddinglayer disposed over the waveguide carrying substrate. An example of sucha device is schematically illustrated in FIG. 1. As shown in both a topview (Panel I) and side view (Panel II, A and B), the arrayed waveguide100 includes a base substrate 102 that has a first refractive index.Portions of the substrate 104-116 are provided having a higherrefractive index, thus allowing them to confine and propagate lightintroduced into them, functioning as waveguide cores. Typically, thesearrayed waveguides receive light from a first optical fiber 120, that isoptically coupled to the waveguides, e.g., connected such that light istransmitted from one to the other, and propagate (via waveguide 104) andoptionally divide the light equally among the waveguides 106-116, whichdivided light is then propagated into optical fibers 122-132. As notedabove, and with reference to panel II, both A and B views, the use ofsuch devices in the fiber optic applications typically has necessitatedthe inclusion of a cladding layer 150, disposed over the surface ofsubstrate 102, in order to better confine and/or isolate the lightwithin the waveguides and to minimize propagation losses. While thesubstrates of the invention are preferably planar substrates having thewaveguide(s) disposed therein, it will be appreciated that for certainapplications, non-planar substrates may be employed, including forexample, fiber based substrates, shaped substrates, and the like.

In contrast to the above described waveguide arrays, the presentinvention exploits the underlying waveguide substrate and waveguides,but eliminates the cladding layer that blocks access to the light thatemanates from the waveguide, thus exposing the substrate surface andproviding more proximal access to the evanescent light field emanatingfrom the waveguides at the substrate surface. As noted previously, alarge number of analytical operations can benefit from the ability tocontrollably illuminate materials at or near a surface and/or at anumber of locations thereon, without excessively illuminating thesurrounding environment. Further, although discussed in preferredaspects as providing access to the evanescent light from an array, itwill be appreciated that modifications of waveguides to enhancedirection of light propagating therethrough, toward the surface, arealso contemplated by the invention, including, e.g., embedded gratingsor other optical components embedded in the waveguide that direct lighttoward the surface region of interest on a substrate. Notwithstandingthe foregoing, exploitation of the evanescent field is preferred as itgives rise to a desired limited illumination volume proximal to thesurface of the substrate for a number of applications.

An example of the devices (also referred to herein as analyticaldevices) of the invention is schematically illustrated in FIG. 2. Aswith FIG. 1, the array 200 includes a substrate 202 in which aredisposed an array of waveguides 206-216 optically coupled to anoriginating waveguide 204. For purposes of the present disclosure, anoriginating waveguide generally refers to a waveguide which is opticallycoupled to more than one additional waveguide, or in some cases, awaveguide that is provided in a substantially different configuration,e.g., a different substrate, a different spatial plane, or having asubstantially different cross-section, refractive index, or path/shapeconfiguration, or the like.

In the absence of a cladding layer, e.g., cladding layer 150 in FIG. 1,the waveguide cores are exposed at the surface 222 of the substrate, orare sufficiently proximal to that surface that the evanescent fieldemanating from the cores extends beyond the surface 222 of thesubstrate. For purposes of the invention, a waveguide that is referredto as being disposed upon or within the surface of a substrateencompasses waveguides that are disposed on but above the surface,within the substrate but at or exposed to the surface, or are disposedwithin the substrate, but sufficiently proximal to the surface that theevanescent wave from light passing through the waveguide still reachesabove the surface to provide an illumination volume.

As noted previously, this provides access to the evanescent lightoutside of each waveguide core. By providing materials at or proximal tothe surface, e.g., particle 224, one can controllably illuminate suchmaterials without illuminating any materials outside of the evanescentfield. Such selective illumination allows for illumination of individualor relatively small numbers of particles, molecules or cells in moreconcentrated solutions of such materials, as described in greater detailbelow. As will be appreciated, controllable illumination includes notonly control of illumination in the axis orthogonal to the substrate orcore surface (z axis), but also in at least one of the axes of the planeof the substrate surface (x or y axes). This lateral control ofillumination, particularly when coupled with additional lateral controlof analyte location, e.g., through the use of immobilization, or otherspatial confinement techniques, e.g., structural barriers, etc.,provides additional advantages of selectivity of observation.

In accordance with the present invention, the substrates may include avariety of different configurations, depending upon the desiredapplication(s). By way of example and as shown in FIG. 3A, a waveguidearray 300 may include a substrate 302 having two or more originatingwaveguides 304 and 354 that are both in optical communication with thearray of waveguides 306-316. Light sources having differentcharacteristics, e.g., different spectral characteristics, frequencies,or the like, are directed into the separate originating waveguides (asshown by arrows 356 and 358), e.g., through a coupled optical fiber, inorder to deliver illumination of different characteristics to the sameset of waveguides 306-316, and consequently surface of the substrate. Analternative configuration is shown in FIG. 3B, where the two or morelight sources provide light to two or more originating waveguides at thesame end of the array substrate, and each originating waveguide is inoptical communication with the same set or overlaps with at least aportion of the same set of waveguides in the array.

Alternatively, individual waveguide array substrates may includemultiple originating waveguides, e.g., like originating waveguides 104and 304 from FIGS. 1 and 3, respectively, that are each coupled toseparate arrays of waveguides, in order to provide for highermultiplexing capabilities of each substrate, including performance ofdifferent analyses on a single substrate, e.g., using different lightsources having differing characteristics.

The arrays may include parallel waveguides, e.g., as shown, or mayinclude patterned waveguides that have a variety of differentconfigurations, including serpentine waveguides, divergent waveguides,convergent waveguides or any of a variety of configurations dependingupon the desired application. For example, where it is desired toprovide evanescent illumination to larger areas of the substrate, it maybe desirable to provide such serpentine waveguides, wider or slabwaveguide(s), or alternatively and likely preferably, larger numbers ofparallel or similarly situated waveguides. As noted previously, thewaveguide substrates may include a single waveguide that may span afraction of the width of the substrate or substantially all of thatwidth. In accordance with preferred aspects however, waveguide arraysare used to split individual originating beams into two or morewaveguides, preferably more than 10 waveguides, more than 20 waveguides,more than 40 waveguides, and in some cases more than 50 waveguides oreven more than 100, 1000, 5000 or more waveguides. The number ofwaveguides may typically vary greatly depending upon the size of thesubstrate used, and the optical resolution of the detection system,e.g., its ability to distinguish materials proximal to differentwaveguides.

The waveguides may individually vary in the size of the core region inorder to vary the evanescent field that one can access. Typically, thewaveguides will have a cross sectional dimension of from about 0.1 toabout 10 μm, preferably from about 0.2 to about 2 μm and more preferablyfrom about 0.3 to about 0.6 μm. A variety of other waveguide dimensionsmay be employed as well, depending upon the desired application. Forexample, in some cases, a single waveguide may be used where thecross-sectional dimension of the waveguide is substantially the same asthe substrate width or length, e.g., a single waveguide thatsubstantially spans a substrate's width. Notwithstanding the foregoing,preferred aspects will provide arrayed waveguides, e.g., multiplewaveguides typically arranged in parallel linear format.

Although described above in terms of a particular type of waveguidestructure, e.g., an embedded waveguide structure, a variety of differentwaveguide structures are exploitable in the present invention, and areshown in FIG. 4A. In particular, the waveguide arrays of the inventionmay employ an embedded waveguide, e.g., as described above and shown inPanel I, and channel waveguides (Panel II and III). FIG. 4B provides aschematic illustration of the general shape of the evanescent field thatwould be yielded by each type of waveguide in FIG. 4A, Panels I-III,respectively (See, e.g., Saleh and Teich, Fundamentals of Photonics,(John Wiley and Sons, 1991) and particularly Chap. 7.2). For purposes ofthe present disclosure, the waveguides of the invention in which thecore is exposed to or proximal to the substrate surface such that theevanescent field emanating form the core extends above the substratesurface, are generally referred to as being disposed upon the surface ofthe substrate, regardless of whether they extend the nominal surface ofthe substrate or are embedded therein, or even embedded thereunder, tosome degree. Thus, for example, all of the waveguide configurationsillustrated in FIGS. 4A and B are generally referred to as beingdisposed upon the surface of the substrate while for certain aspects,e.g., embedded waveguides, the core of the waveguide may additionally bereferred to as being disposed within the surface of the substrate.

In some cases, the waveguides described herein are generally producedusing conventional ion implantation techniques to selectively ion dopeselected regions of SiO₂ substrates to provide patterned regions ofhigher refractive index, so as to function as waveguides embedded in theunderlying substrate. Examples of such devices are disclosed in, e.g.,Marcuse, Theory of Dielectric Optical Waveguides. Second Ed. (AcademicPress 1991). Alternate waveguide fabrication processes andconfigurations are equally applicable to the present invention,including hybrid material waveguides, e.g., employing polymericmaterials as a portion or all of the subject substrate, e.g., a polymercore having a first refractive index, disposed within a substrate ofanother material having a second refractive index, which may bepolymeric, or another material, e.g., glass, quartz, etc.

Additional optical functionalities may be provided upon or within thesubstrates of the invention, including, e.g., providing additionaloptical confinements upon the substrate, such as zero mode waveguides asdiscussed in U.S. Pat. Nos. 6,991,726 and 7,013,054, previouslyincorporated herein by reference. Other optical functionalities that maybe integrated into or upon the substrates including, e.g., mask layers,lenses, filters, gratings, antireflective coatings, or the like. Otherfunctionalities may be incorporated into the fabricated substrate thatoperate on and/or in conjunction with the waveguides or waveguide arraysof the invention. For example, optical switching or attenuationcomponents may be provided upon or within the substrates of theinvention to selectively direct and/or modulate the light passingthrough a given waveguide or waveguides.

By way of example, the waveguide array may have a controllable opticalswitch or attenuator built into its structure which can provide controlover the amount of light allowed to enter the waveguide structure. Suchcontrol permits the careful selection of optimal light levels for agiven analysis being carried out using the substrate. Further, usingindependently activated switches or attenuators on a waveguide arraypermits one to independently control light application to one or asubset of waveguides in an array. In still other advantageousapplications, in combination with multiple light sources coupled to thesame waveguides through different originating waveguides, opticalswitching of the input waveguides will permit one the ability to selectthe light source(s) for any subset or all of the waveguides at a giventime, or even to modulate the intensity of a light from a given source,on the fly. By controlling the light from individual sources, e.g.,where such sources have differing spectra, one can consequently controlthe wavelength of the light reaching a given waveguide and itsassociated reaction regions.

In addition to the ability to more precisely control the lightparameters of the overall system for precise tuning of the application,the controlled aspect of the light application provides furtherabilities to mitigate potential adverse effects of excessiveillumination on reaction components, such as photo-damage effects onreactants, or other reaction components resulting from prolonged highintensity illumination.

A variety of different optical devices may be employed in controllinglight passage through the waveguides used in the substrates of theinvention. In particular, optical modulators, such as Mach-Zhendermodulators (see, e.g., U.S. Pat. No. 7,075,695 for discussion of highspeed Mach-Zhender modulators), Michelson modulators, thermally tunablemodulators that may include other optical devices (see, e.g., PublishedU.S. Patent Application No. 2005-0232543 for a discussion of thermallytunable modulators) or may employ heating elements to modulate therefractive index of one or more waveguides, optical switches, and thelike.

FIG. 5 schematically illustrates the use of optical modulators in thecontext of the waveguide array based substrates of the invention. Asshown, a substrate 502 is provided including a number of opticalwaveguides, e.g., surface exposed waveguides 504, 506 and 508. Thewaveguides 504-508 are optically coupled to a light source (not shown)via optical fiber 510 and input waveguide 512. Each of waveguides504-508 includes a Mach-Zhender modulator, as shown by electrode pairs520-524, respectively, and the associated branch waveguides (514-518,respectively). Although shown having dual electrode control, singleelectrode modulators may be employed (and in certain preferred aspects,are employed) to modulate the refractive index of one path to adjust thephase of light traveling therethrough. In particular, rather thanelectrode pair 520 being disposed over both channels of branch waveguide514, only a single electrode would be disposed over one of the branchwaveguides to modulate the phase of light passing therethrough.Similarly, in the context of interferometers employing thermal controlrather than electric fields, it will be appreciated that a singleheating element, e.g., taking the place of a single electrode, willtypically be used. Preferred heating elements include resistive heatersdisposed over the waveguide, e.g., patterned electrodes having highresistivity over the branch waveguide. Other heating elements maylikewise be employed, including, e.g., infrared heating elements,pettier devices, and the like.

In addition to the optical functionalities of the substrates of theinvention, in some cases, such substrates may include additionalfunctionalities that provide a defined region on the substrate surfaceto limit the access that reagents or other elements have to theillumination zone above a waveguide. For example, in some cases, thesubstrates may include a patterned structure or set of structures overthe surface of the substrate providing selected exposure of the surfaceexposed waveguide(s). Such selected regions may provide limited areas ofillumination on a given substrate by blocking the illumination regionexisting above other portions of the waveguide(s). As a result, onlyselected portions of the surface will be within the illumination regionof the waveguides. Such regions may be selected to align with detectionsystems or the requirements of such systems, e.g., sample spacingpermitting spectral separation of signals from each region (See, e.g.,U.S. patent application Ser. No. 11/704,733, filed Feb. 9, 2007, whichis incorporated herein by reference in its entirety for all purposes).In addition to limited access, such structures may also providestructural confinement of reactions or their components, such as wellsor channels. In one aspect, for example, microfluidic channels may beprovided disposed over surface exposed waveguide or waveguide array.Such channels may be independently used to deliver different reagents todifferent portions of a waveguide or waveguide array.

FIG. 6 provides a schematic illustration of the structure of exemplarysubstrates according to this aspect of the invention. As shown in FIG.6A, a first substrate 600 includes a waveguide 602 at or sufficientlyproximal to the surface of the underlying substrate 600 that some of theevanescent wave from the waveguide 602 can reach above that surface. Amask layer 604 is provided over the underlying substrate that maskscertain portions 606 of the waveguide but not other portions 608, thatremain accessible to materials disposed over the overall substrate. Inparticular, the evanescent wave from exposed waveguide region 608 canreach reagents deposited over the surface of the overall substrate, andparticularly within wells 610. By virtue of mask layer 604, theevanescent wave from the other blocked portions of the waveguide 606,will not reach any materials deposited over the surface of thesubstrate. As a result, one can pre-select those regions that areoptically interrogatable, and thus direct optical systems appropriately.A top view of the overall substrate is shown in FIG. 6B, where the wells610 are provided through the mask layer to expose portions of theunderlying waveguides 602.

Substrates including mask layer 604 may be prepared by a variety ofknown fabrication techniques. For example, lithographic techniques maybe used to define the mask layer out of polymeric materials, such asphotoresists, using e.g., conventional photolithography, e-beamlithography, or the like. Alternatively, lithographic techniques may beapplied in conjunction with layer deposition methods to deposit metalmask layers, e.g., using aluminum, gold, platinum, chrome, or otherconventionally used metals, or other inorganic mask layers, e.g., silicabased substrates such as silicon, SiO₂, or the like. In particularlypreferred aspects, both the underlying substrate and the mask layer arefabricated from the same material, which in particularly preferredaspects, is a transparent substrate material such as an SiO₂ basedsubstrate such as glass, quartz, or fused silica. By providing the maskand underlying layers of the same material, one can ensure that the twolayers have the same interactivity with the environments to which theyare exposed, and thus minimize any hybrid surface interactions.

In the case of SiO₂ based substrates and mask layers, conventionalfabrication processes may be employed. In particular, a glass substratebearing the surface exposed waveguide has a layer of resist depositedover its surface. A negative of the mask layer is then defined byappropriate exposure and development of the resist layer to provideresist islands where one wishes to retain access to the underlyingwaveguide. The mask layer is then deposited over the surface and theremaining resist islands are removed, e.g., through a lift off process,to provide the openings to the underlying waveguides. In the case ofmetal layers, deposition may be accomplished through a number of means,including evaporation, sputtering or the like. Such processes aredescribed in, e.g., U.S. Pat. No. 7,170,050, which is incorporatedherein by reference in its entirety for all purposes. In the case ofsilica based mask layers, a CVD process may be employed to deposit asilicon layer onto the surface. Following lift off of the resist layer,a thermal oxidation process can convert the mask layer to SiO₂.Alternatively, etching methods may be used to etch access points tounderlying waveguides using conventional processes. For example, asilicon layer may be deposited over an underlying waveguide substrate. Aresist layer is then deposited over the surface of the silicon layer andexposed and developed to define the pattern of the mask. The accesspoints are then etched from the silicon layer using an appropriatedifferential etch to remove silicon but not the underlying SiO₂substrate. Once the mask layer is defined, the silicon layer is againconverted to SiO₂ using, e.g., a thermal oxidation process.

In addition to the advantages of reduced auto-fluorescence, waveguidesubstrates having an integrated mask layer provide advantages of opticalalignment over similar arrays of wells or structures. In particular, inilluminating an ordered array of reaction regions with minimal excessillumination, one typically presents a corresponding array ofillumination spots. In doing so, one must take substantial care inaligning the optical presentation of the illumination spots to theordered array of reaction regions. Such alignment presents challenges ofboth design and robustness, as such systems may be prone to drifting orother misalignment influences. Where, as in the present invention,illumination is hard wired into the substrate by virtue of theintegrated waveguide, alignment is automatic.

In other cases, surface features may include other confinementstrategies, including, e.g., chemical surface functionalities that areuseful in a variety of surface analytical operations, such ashydrophobic coatings or hydrophilic coatings that are optionallypatterned, to provide confinement or direction to aqueous materials,chemical derivatization, e.g., to facilitate coupling of otherfunctional groups or otherwise, e.g., by providing hydrophobic barrierspartially or completely surrounding a desired region. As will beappreciated, in some cases, particularly where structural confinement isprovided upon the surface of the substrate, it may not be necessary todivide up light through a series of discrete waveguides in a givensubstrate. In particular, because one can obtain a desired level ofmultiplex and spatial separation organization from structurally dividingup the surface, one need not obtain that property through the use ofseparate waveguides. In such cases, a single field waveguide disposed atthe surface of the substrate will suffice to deliver light to thevarious reaction regions on the surface of the substrate, e.g., asdefined by the mask layer. An example of this is illustrated in FIG. 6C,where the waveguide 602 is shown (shaded) as extending across the entiresurface area of the substrate 600 and the wells or apertures 610 throughthe mask layer, leave exposed portions of the waveguide 608 to definethe access points to the evanescent wave coming from the waveguide.

In addition to structures and strategies that provide for positioningand/or confinement upon a substrate surface, other components may beprovided upon a substrate, including backside coatings for thesubstrate, e.g., antireflective coatings, optical indicator components,e.g., structures, marks, etc. for the positioning and or alignment ofthe substrate, its constituent waveguides, and/or for alignment of othercomponents. Other components may include substrate packaging components,e.g., that provide fluidic interfaces with the substrate surface, suchas flow cells, wells or recesses, channel networks, or the like, asmacrostructures as compared to the surface defined structures above, aswell as alignment structures and casings that provide structuralprotection for the underlying substrates and interactive functionalitybetween the substrates and instrument systems that work with/analyze thesubstrates.

III. Methods and Applications

A. Generally

As noted previously, the substrates, systems and methods of theinvention are broadly applicable to a wide variety of analyticalmethods. In particular, the substrates of the invention may be employedin the illumination mediated analysis of a range of materials that aredisposed upon or proximal to the substrate's surface. Such analysesinclude, inter alia, a number of highly valued chemical, biochemical andbiological analyses, including nucleic acid analysis, proteininteraction analysis, cellular biology analysis, and the like.

B. Exemplary Applications

1. Sequencing by Synthesis

One example of an analytical operation in which the present invention isparticularly applicable is in the determination of nucleic acid sequenceinformation using sequence by synthesis processes. Briefly, sequencingby synthesis exploits the template directed synthesis of nascent DNAstrands, e.g., using polymerase mediated strand extension, and monitorsthe addition of individual bases to that nascent strand. By identifyingeach added base, one can deduce the complementary sequence that is thesequence of the template nucleic acid strand. A number of “sequence bysynthesis” strategies have been described, including pyrosequencingmethods that detect the evolution of pyrophosphate upon theincorporation of a given base into the nascent strand using aluminescent luciferase enzyme system as the indicating event. Becausethe indicator system is generic for all four bases, the process requiresthat the polymerase/template/primer complex be interrogated with onlyone base at a time.

Other reported sequence by synthesis methods employ uniquely labelednucleotides or nucleotide analogs that provide both an indication ofincorporation of a base, as well as indicate the identity of the base(See, e.g., U.S. Pat. No. 6,787,308). Briefly, these methods employ asimilar template/primer/polymerase complex, typically immobilized upon asolid support, such as a planar or other substrate, and interrogate itwith nucleotides or nucleotide analogs that may include all four bases,but where each type of base bears an optically detectable label that isdistinguishable from the other bases. These systems employ terminatorbases, e.g., bases that, upon incorporation, prevent further strandextension by the polymerase. Once the complex is interrogated with abase or mixture of bases, the complex is washed to remove anynon-incorporated bases. The washed extended complex is then analyzedusing, e.g., four color fluorescent detection systems, to identify whichbase was incorporated in the process. Following additional processing toremove the terminating group. e.g., using photochemistry, and in manycases, the detectable label, the process is repeated to identify thenext base in the sequence. In some cases, the immobilized complex isprovided upon the surface as a group of substantially identicalcomplexes, e.g., having the same primer and template sequence, such thatthe template mediated extension results in extension of a large numberof identical molecules in a substantially identical fashion, on a stepwise basis. In other strategies, complexes are immobilized in a way thatallows observation of individual complexes resulting in a monitoring ofthe activity of individual polymerases against individual templates.

As will be appreciated, immobilization or deposition of thepolymerase/template/primer complex upon or proximal to the surface ofthe waveguide core in the waveguide arrays of the invention will allowillumination, and more notably in the case of fluorescence based assays,excitation, at or near selected regions of the surface without excessiveactivation and fluorescence interference from the surroundingenvironment, which can be a source of significant noise in fluorescencebased systems.

In another sequencing-by-synthesis process, one monitors the stepwiseaddition of differently labeled nucleotides as they are added to thenascent strand and without the use of terminator chemistries. Further,rather than through a one-base-at-a-time addition strategy, monitoringof the incorporation of bases is done in real time, e.g., without theneed for any intervening wash steps, deprotection steps or separatede-labeling steps. Such processes typically rely upon optical strategiesthat illuminate and detect fluorescence from confined reaction volume;such that individual complexes are observed without excessiveinterference from labeled bases in solution that are not beingincorporated (See U.S. Pat. Nos. 6,991,726 and 7,013,054, previouslyincorporated herein, and U.S. Pat. Nos. 7,052,847, 7,033,764, 7,056,661,and 7,056,676, the full disclosures of which are incorporated herein byreference in its entirety for all purposes), or upon labeling strategiesthat provide fluorescent signals that are indicative of the actualincorporation event, using, e.g., FRET dye pair members on a base and ona polymerase or template/primer (See U.S. Pat. Nos. 7,052,847, 7033,764,7,056,661, and 7,056,676, supra).

This aspect of the invention is schematically illustrated in FIG. 7. Asshown in panel I, a portion of a substrate surface 702 that includes awaveguide 704 as described herein, is provided with immobilizedcomplexes 706 and 708 of the template nucleic acid sequence, a primersequence and a polymerase enzyme. The illumination volume resulting fromevanescent field emanating from the light propagating down the waveguideprovides a relatively small volume in which fluorescent compounds willbe excited, as shown by dashed field line 710. As a result, only thosecomplexes sufficiently close to the waveguide core, e.g., complex 706,will be excited, and those outside this volume, e.g., complex 708, willnot be illuminated. In the context of real time sequencing methods, thecomplex is interrogated with a mixture of all four, distinguishablylabeled nucleotide analogs, e.g., nucleotides 712 (A, T, G, C) (seepanel II). Upon incorporation (Panel III), a given nucleotide, e.g., A,will be retained within the illumination volume for a period longer thanthat which would occur based upon normal diffusion of bases into and outof the illumination volume, and as such is identifiable as anincorporated base. Bases in solution or not incorporated, e.g., T and G,or incorporated by non-illuminated complex, e.g., C, will not beilluminated and will therefore not be detected. By monitoringincorporation as it progresses, one can identify with reasonably highaccuracy, the underlying template sequence. While a variety of methodsmay be employed, preferred methods of monitoring the reactions as theyoccur at the surface (or in the case of step-wise methods after theyoccur), is accomplished using detection systems as described elsewhere,herein. Although described in one exemplary application as being usefulin real-time sequencing applications, it will be appreciated that thesubstrates methods and systems of the invention are equally applicableto the other sequence by synthesis applications described herein thatemploy illumination based activation of signaling or labeling mechanism,e.g., fluorescence based systems.

In accordance with the foregoing sequence by synthesis methods, one mayoptionally provide the complexes over an entire surface of a substrate,or one may selectively pattern the immobilized complexes upon orproximal to the waveguide cores. Patterning of complexes may beaccomplished in a number of ways using selectively patternable chemicallinking groups, and/or selective removal or ablation of complexes not inthe desired regions. In some cases, one can employ the waveguides inselectively patterning such complexes using photoactivatable chemistrieswithin the illumination region. Such strategies are described in detailin U.S. patent application Ser. No. 11/394,352 filed Mar. 30, 2006, andincorporated herein by reference in its entirety for all purposes.

In addition to selective immobilization, and as noted above, in somecases it is desirable to immobilize the complexes such that individualcomplexes may be optically resolvable, e.g., distinguished from othercomplexes. In such cases, the complexes may be immobilized using highlydilute solutions, e.g., having low concentrations of the portion of thecomplex that is to be immobilized, e.g., the template sequence, thepolymerase or the primer. Alternatively, the surface activation forcoupling of the complex component(s) may be carried out to provide a lowdensity active surface to which the complex will be bound. Such surfaceshave been described in U.S. patent application Ser. No. 11/240,662,filed Sep. 30, 2005, which is incorporated herein by reference in itsentirety for all purposes. Again, such low density complexes may bepatterned just upon or proximal to the waveguides or they may beprovided across the surface of the substrate, as only those reactioncomplexes that are proximal to the waveguides will yield fluorescentsignals.

While described in terms of real-time nucleic acid sequencing bysynthesis, it will be appreciated that a wide variety of real-time,fluorescence based assays may be enhanced using the substrates, methodsand systems of the invention.

Molecular Arrays and other Surface Associated Assays

Another exemplary application of the substrates and systems of theinvention is in molecular array systems. Such array systems typicallyemploy a number of immobilized binding agents that are each specific fora different binding partner. The different binding agents areimmobilized in different known or readily determinable locations on asubstrate. When a fluorescently labeled material is challenged againstthe array, the location to which the fluorescently labeled materialbinds is indicative of it's identity. This may be used inprotein-protein interactions, e.g., antibody/antigen, receptor-ligandinteractions, chemical interactions, or more commonly e.g., nucleic acidhybridization interactions. See, U.S. Pat. Nos. 5,143,854, 5,405,783 andrelated patents, and GeneChip® systems from Affymetrix, Inc.

In accordance with the application of the invention to arrays, a numberof binding regions, e.g., populated by known groups of nucleic acidprobes, are provided upon a substrate surface upon or proximal to thewaveguides such that a hybridized fluorescently labeled probe will fallwithin the illumination region of the waveguide. By providing forselective illumination at or near the surface, one can analyzehybridized probes without excessive interference from unboundfluorescent materials.

This aspect of the invention is schematically illustrated in FIG. 8. Asshown, a substrate surface 802 is provided with groups of molecules,e.g., nucleic acid probes 804, 806 and 808, where each probe group has adifferent binding specificity, e.g., to different complementary nucleicacid sequences. The groups are each provided upon or sufficientlyproximal to a waveguide core, e.g., waveguide cores 810, 812, and 814,respectively, so that their respective illumination volumes, indicatedby the dashed lines 816, encompasses a hybridized, fluorescently labeledprobe 818. Illumination of the probe then excites the fluorescent label820 allowing observation of hybridization. Such observation may becarried out post reaction, or in some cases as desired, in real time.

3. Cellular Observation and Analysis

In still another exemplary application, cell based assays or analysesmay be carried out by providing cells adhered to the substrate surfaceover the waveguides. As a result, one could directly monitorfluorescently labeled biological functions, e.g., the uptake offluorescent components, the generation of fluorescent products fromfluorogenic substrates, the binding of fluorescent materials to cellcomponents, e.g., surface or other membrane coupled receptors, or thelike.

4. Other Applications

It will be appreciated by those of ordinary skill that the substrates ofthe invention may be broadly applicable in a wider variety ofapplications that analytical processes. For example, such substrates andmethods may be employed in the identification of location of materialson surfaces, the interrogation of quality of a given process providedupon the surface, the photo manipulation of surface bound materials,e.g., photo-activation, photo-conversion and/or photo-ablation. As such,while some of the most preferred applications of the present inventionrelate to analytical operations and particularly hi the fields ofchemistry, biochemistry, molecular biology and biology, the discussionof such applications in no way limits the broad applicability of theinvention.

IV. Systems

In general, the substrates of the invention are employed hi the contextof other components as a system or one or more subsystems. By way ofexample, in preferred aspects, the substrates of the invention areemployed in the analysis of materials disposed upon the substratesurface as described elsewhere herein. In such cases, the substrates ofthe invention are generally exploited in conjunction with and as a partof an analytical substrate and reagent system that is used in thedesired analysis. Such reagent systems may include proteins, such asenzymes and antibodies, nucleic acids including nucleotides,nucleosides, oligonucleotides and larger polymers of same, substratesfor a given reaction, cells, viruses or phages, or any of a variety ofdifferent chemical, biochemical or biological components for a desiredanalysis.

In addition to the “wet-ware” components of the systems set forthinitially above, the invention also includes the substrates of theinvention in conjunction with hardware and/or software systemcomponents. As noted previously, such hardware components include, e.g.,optical components such as lenses, mirrors, prisms, illumination sourcesand the like, detection systems, while software components includeprocesses for controlling overall systems and/or software forprocessing, evaluating and presenting data derived from those systems.

A. Reagent Systems and Kits

As set forth above, the substrates of the invention may be appliedand/or packaged in kits with other reagents, buffers and other adjunctsthat are used in the desired analysis. The nature of such reagents isgenerally application specific and will vary according to suchapplications. By way of example in application of such substrates tonucleic acid sequencing methods, as described below, the substrates mayinclude one or more of template nucleic acids, nucleic acid probes,polymerase enzymes, native and/or nonnative nucleotides or nucleotideanalogs, that will in certain preferred aspects, include labeling groupssuch as fluorescent labels. One or more of the foregoing components maybe either packaged with and/or applied as immobilized components on thesurface of the substrate that is exposed to the evanescent radiationfrom the waveguides.

In the context of application in real time sequencing by synthesis,reagent systems may include polymerase enzymes, as described in U.S.Patent Application No. 60/753,515 filed Dec. 22, 2005, which isincorporated herein by reference, or commercially available polymeraseenzymes, e.g., taq polymerases. Sequanase® polymerases, Phi 29polymerases, DNA Polymerase I, Klenow fragment, or any of a variety ofother useful polymerases known to those of skill in the art.Additionally, such systems may include primer sequences that are eitherspecific for a particular template sequence or are specific for auniversal tag sequence that may be ligated to or otherwise provided inline with the template sequence. Such systems may further includenucleotides or nucleotide analogs, such as fluorescently labelednucleotides like those described in U.S. patent application Ser. No.11/241,809, filed Sep. 29, 2005, Published U.S. Application No.2003/0124576, and U.S. Pat. No. 6,399,335, the full disclosures of whichare all incorporated herein by reference for all purposes.

Optics and Instrumentation

As noted elsewhere herein, instrument systems are also included for usein conjunction with the substrates and methods of the invention.Typically, such systems include the substrates of the inventioninterfaced with appropriate light sources, such as one or more lasers,for delivering desired electromagnetic radiation through the waveguides.Also included is an appropriate optical train for collecting signalsemanating from the substrate surface following illumination, anddetection and data processing components for detecting, storing andpresenting signal information.

One example of a system for use in the present invention is illustratedin FIG. 9. As shown, the system 900 includes a substrate of theinvention 902. Laser 904 and optional additional laser 906 are opticallycoupled to the waveguide array within the substrate, e.g., via opticalfibers. An optical train is positioned to receive optical signals fromthe substrate and typically includes an objective 910, and a number ofadditional optical components used in the direction, filtering, focusingand separation of optical signals. As shown, the optical train includesa wedge prism for separating spectrally different signal components, anda focusing lens 914 that images the signal components upon an arraydetector, e.g., EMCCD 916. The detector is then operatively coupled to adata storage and processing system, such as computer 918 for processingand storage of the signal data and presentation of the data in a userdesired format, e.g., on printer 920. As will be appreciated, a numberof other components may be included in the systems described herein,including optical filters for filtering background illumination orbleed-through illumination from the light sources, from the actualoptical signals. Additionally, alternate optical trains may employcascaded spectral filters in separating different spectral signalcomponents.

While illustrated with a first light source, e.g., laser 904, and anoptional second light source, e.g., optional laser 906, it will beappreciated that additional light sources may be provided opticallycoupled to the waveguide arrays, e.g., using additional originatingwaveguides to direct light from each the various sources to all or asubset of the waveguides in a given array. For example, in some cases, 3light sources, 4 light sources or more may be used. Additional lightsources will preferably provide light having different spectralcharacteristics, e.g., peak wavelengths, to the waveguides, althoughthey may also be employed to provide additional intensity or variationsin other light characteristics, such as frequency,

The detection system is typically configured to detect signals fromlarge areas of the substrate, e.g., multiple signals emanating from aplurality of different regions on the substrate, and preferably, do sosimultaneously. Thus while scanning detection optics may be employed forcertain applications of the invention, in general, larger area imagingdetection systems are preferred.

Other optical trains and detection systems that may be employed in thepresent invention are described in U.S. patent application Ser. No.11/201,768 (filed Aug. 11, 2005) and 60/772,908 (filed Feb. 13, 2006),each of which is incorporated herein by reference, which, with provisionof a waveguide substrate of the invention and direction of excitationillumination through the waveguides, would be directly useful in thecontext of the invention. In particular, in certain aspects, the imagedsignal will be a series of discrete signal sources or points of signalorigin on the overall surface of the substrate. As such, the detectionsystems described in the aforementioned application would be directlyapplicable to the present invention.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. To the extent not already expressly incorporatedherein, all published references and patent documents referred to inthis disclosure are incorporated herein by reference in their entiretyfor all purposes.

What is claimed is:
 1. An analytical device, comprising: a substrate comprising a first surface and at least a first optical waveguide disposed upon the first surface; an optically resolvable molecular complex immobilized within a confined region and sufficiently proximal to the first surface to be illuminated by an evanescent field emanating from the optical waveguide when light is passed through said optical waveguide; and at least a first excitation radiation source optically coupled to the substrate to provide the evanescent field by passing excitation light into the optical waveguide.
 2. The device of claim 1, wherein a core of the optical waveguide is exposed at the first surface of the substrate in the confined region.
 3. The device of claim 1, further comprising at least one filter integrated into the substrate.
 4. The device of claim 1, further comprising at least one grating integrated into the substrate.
 5. The device of claim 1, further comprising a mask layer disposed over the first surface of the substrate, wherein the mask layer comprises an aperture disposed therethrough, the aperture providing the confined region within which the optically-resolvable molecular complexes is immobilized.
 6. The device of claim 5, wherein portions of the mask layer lacking the apertures block the evanescent field emanating from the array.
 7. The device of claim 5, wherein the apertures are optical confinements.
 8. The device of claim 1, wherein the optically resolvable molecular complex comprises an enzyme.
 9. The device of claim 8, wherein the enzyme is a polymerase.
 10. The device of claim 1, wherein the optically resolvable molecular complex comprises an individual analyte comprising a fluorescent or fluorogenic label that emits a signal in response to the excitation light.
 11. A method of detecting a signal from an analyte, comprising: providing a substrate comprising a) a first surface; b) at least a first optical waveguide proximal to the first surface; c) a plurality of optically resolvable molecular complexes immobilized within confined regions and sufficiently proximal to the first optical waveguide to be illuminated by an evanescent field emanating from the first optical waveguide when light is passed through said optical waveguide; exposing the first surface to an analyte comprising a fluorescent or fluorogenic moiety that emits a signal in response to the evanescent field; directing light through the first optical waveguide such that the evanescent field from the waveguide illuminates the optically resolvable molecular complexes; and upon interaction of the analyte with one of the optically resolvable molecular complexes, detecting a signal from the analyte.
 12. The method of claim 11, wherein the confined regions are within apertures in a mask layer disposed over the first surface.
 13. The method of claim 12, wherein the mask layer blocks the evanescent field above portions of the substrate that are not disposed beneath one of the apertures.
 14. The method of claim 12, wherein the apertures are optical confinements.
 15. The method of claim 11, wherein each of the optically resolvable molecular complexes comprises a polymerase enzyme in a complex with a target nucleic acid and a primer complementary to a portion of the target nucleic acid; and wherein the analyte is a nucleotide or nucleotide analog.
 16. The method of claim 11, wherein the substrate further comprises at least one grating integrated into the substrate.
 17. The method of claim 11, wherein the substrate further comprises at least one filter integrated into the substrate.
 18. The method of claim 11, wherein the first optical waveguide is exposed at the first surface of the substrate within the confined regions.
 19. The method of claim 11, wherein the fluorescent or fluorogenic moiety is removed from the analyte during the interaction.
 20. A method of determining a nucleotide sequence of a template nucleic acid, the method comprising: providing a substrate comprising a first surface and at least a first optical waveguide proximal to the first surface of the substrate; immobilizing an optically resolvable polymerase enzyme in a confined region sufficiently proximal to the first optical waveguide to be illuminated by an evanescent field emanating from the first optical waveguide, wherein the polymerase enzyme is further bound to the template nucleic acid, which is further bound to a primer; directing light through the first optical waveguide such that the evanescent field from the waveguide illuminates the confined region; incorporating a plurality of nucleotides into a nascent strand complementary to the template nucleic acid using template-directed synthesis catalyzed by the polymerase enzyme, wherein for each of the nucleotides incorporated into the nascent strand, an identity of a base comprised therein is determined by detecting a signal produced by said each of the nucleotides when exposed to the evanescent field, thereby determining a nucleotide sequence of the nascent strand; and determining a complementary nucleotide sequence that is complementary to the nucleotide sequence of the nascent strand, wherein the complementary nucleotide sequence is the nucleotide sequence of the template nucleic acid.
 21. The method of claim 20, wherein a plurality of optically resolvable polymerase enzymes are immobilized in a plurality of confined regions.
 22. The method of claim 20, wherein the incorporating is done in real time without the use of terminator chemistries.
 23. The method of claim 20, wherein the substrate further comprises at least one grating integrated into the substrate.
 24. The method of claim 20, wherein the substrate further comprises at least one filter integrated into the substrate.
 25. The method of claim 20, wherein the first optical waveguide is exposed at the first surface of the substrate within the confined region. 